The three isoforms of nonmuscle myosin II (NM II) play a variety of roles during mouse embryonic development, and mutations in two of the genes encoding the heavy chains (MYH9 which encodes NM II-A and MYH14 which encodes NM II-C) are associated with human abnormalities. However point mutations in the gene encoding NM II-B (MYH10) have not been reported. We generated point mutant knock-in mice expressing motor-deficient NM II-B. Homozygous mice die at E14.5 in cardiac failure and exhibit novel abnormalities not seen in NM II-B ablated or NM II-B/II-C doubly ablated mice: a failure in midline fusion resulting in a cleft palate, ectopia cordis, and a large omphalocele, which resembles the human syndrome Pentalogy of Cantrell. Impaired apoptosis of mesenchymal cells in the fusing sternum and endocardial cushions contributes to these abnormalities. Expression of NM II-A and II-B, but not II-C in mesenchymal cells provides evidence that mutant II-B is interfering with wild type NM II-A function. One cardiac abnormality found in both mutant and NM II-B knockout mice relates to the cardiac outflow tract. The failure in myocyte disassociation exhibited by the II-B point mutants contributes to abnormal displacement of the aorta to the right ventricle in mutant mice resulting in both the aorta and pulmonary artery emanating from the same ventricle. The apparent cause is a failure in translocation of actin filaments by the mutant NM II-B motor that is required for disassembly of myocyte cell-cell adhesions. Specific expression of Rho kinase-1 in the fusing sternum and developing outflow tract cardiac myocytes suggests that this kinase mediates phosphorylation of NM II in these cells. Our studies show that activated NM II plays important roles in regulating apoptosis and disassembly of cell-cell adhesions during body wall closure and cardiac outflow tract alignment. They also emphasize the dual roles for NM II, one based on the crosslinking of actin-filaments and the other on translocation of actin-filaments. The failure in ventral wall closure resulting in cardiac externalization and the presence of a large omphalocele in our mutant mice has prompted us to study children with the diagnosis of Pentalogy of Cantrell for mutations in the genes encoding NM II-B and related proteins.